Methods and compositions for analysis of plant gene function

a technology of plant gene function and composition, applied in the field of molecular biology, can solve the problems of large amount of time required to obtain, and no virus is currently available as a vector,

Inactive Publication Date: 2005-02-17
SAMUEL ROBERTS NOBLE FOUND
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  • Summary
  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0009] Thus, in accordance with the present invention, there is provided an isolated nucleic acid sequence comprising RNA 1, RNA 2 and / or RNA 3 of F-BMV, or the complement thereof. The isolated nucleic acid sequence may have RNA 1 comprising a nucleic acid sequence selected from the group consisting of a) a nucleic acid sequence encoding the polypeptide encoded by SEQ ID NO:1; b) a nucleic acid sequence hybridizing to SEQ ID NO:1 under high stringency conditions and encoding a polypeptide having the same biological activity as the polypeptide encoded by SEQ ID NO:1; c) a nucleic acid sequence comprising the nucleic acid sequence of SEQ ID NO:1; and d) a nucleic acid sequence having 1% or less changes as compared to the nucleic acid sequence of SEQ ID NO:1. The isolated nucleic acid sequence may have RNA 2 comprising a nucleic acid sequence selected from the group consisting of a) a nucleic acid sequence encoding the polypeptide encoded by SEQ ID NO:2; b) a nucleic acid sequence hybridizing to SEQ ID NO:2 under high stringency conditions and encoding a polypeptide having the same biological activity as the polypeptide encoded by SEQ ID NO:2; c) a nucleic acid sequence comprising the nucleic acid sequence of SEQ ID NO:2; and d) a nucleic acid sequence having 1% or less changes as compared to the nucleic acid sequence of SEQ ID NO:2. “Having the same biological activity” is defined as being part of a genome that infects rice systemically at 25° C.

Problems solved by technology

However, these techniques require a large amount of time to obtain results.
No virus is currently available as a vector for foreign gene expression in rice (Oryza sativa), a major crop throughout Asia and North America.

Method used

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  • Methods and compositions for analysis of plant gene function
  • Methods and compositions for analysis of plant gene function
  • Methods and compositions for analysis of plant gene function

Examples

Experimental program
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Effect test

example 1

Identification and Isolation of F-BMV

[0051] An uncharacterized virus was isolated from an infected tall fescue plant obtained from a breeder's stock of fescue lines at the University of Missouri and maintained in a greenhouse. The virus was determined to be an isolate of BMV by electron microscopy, western blot assay using an antibody against BMV coat protein and northern blot assay using an RNA profile specific for the conserved 3′ untranslated region of the BMV genomic RNAs. Because this virus infected tall fescue, it was designated F-BMV. R-BMV was from a previously described source and maintained in a growth chamber at the Noble Foundation (Ding et al., 1999).

[0052] An initial study was made of the host range of the virus. Plants of three rice cultivars were inoculated with F-BMV or R-BMV virion and grown inside a greenhouse at 25° C. The inoculated plants were observed for disease symptoms for 28 days and tested for virus infection by immunocapture RT-PCR using an antibody ag...

example 2

Cloning and Sequencing of F-BMV and R-BMV

[0053] Virions of F-BMV and R-BMV (Ding et al., 1999) were purified from systemically infected barley leaves by PEG precipitation and differential centrifugation (Lane, 1981). Viral RNA was then isolated from purified virions through phenol / chloroform extraction and ethanol precipitation. First-strand cDNA of RNAs 1, 2 and 3 representing each virus genome were synthesized by priming viral RNAs with Primer HK-R (5′-GACAATGGTCTCTTTTAGAG-3′) (SEQ ID NO:5). The ten 5′-most nucleotides of the HK-R primer sequence contain a PshA1 restriction site coincident with the 3′ end of the R-BMV RNA sequence. Full-length PCR products of the RNAs 1, 2 and 3 were synthesized for each virus using the HK-R primer and primers containing sequences corresponding to the T3 promoter sequence and the 5′ end of the respective R-BMV RNA sequences: (i.e., HK-1F, 5′-AATTAACCCTCACTAAAGGGAGAGTAGACCACGGAACGAGGT-3′ for RNA 1 (SEQ ID NO:6); HK-2F, 5′-AATTAACCCTCACTAAAGGGAGAGT...

example 3

Production of Infectious Transcripts from cDNAs of F-BMV and R-BMV

[0055] Plasmid DNAs representing RNA 1 of F-BMV (pF1-11) and R-BMV (pR1-26) were linearized with the restriction enzyme SpeI. Plasmid DNAs representing RNA 2 and 3 of the F-BMV (pF2-2 and pF3-5) and R-BMV (pR2-9 and pR3-3) were linearized with the restriction enzyme PshA. In vitro transcripts were synthesized from each clone using the mMeSSAGE mMACHINE transcription kit as described (Ambion, Austin, Tex.). The three RNA transcripts representing their respective viruses (i.e., F-BMV or R-BMV) were equally mixed and inoculated to leaves of Hordeum vulgare (barley) cv. Morex, Chenopodium amaranticolor and C. quinoa (5 to 6 μl mixed RNA transcripts / leaf, one leaf / plant). The inoculated plants were grown inside a growth chamber at 22 / 18° C. (day / night). At 3 to 4 days post inoculation (dpi), C. amaranticolor and C. quinoa leaves showed chlorotic lesions (FIG. 1A-1B). Barley plants inoculated with the transcripts represent...

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Abstract

The invention provides novel methods and compositions for modulating gene function in plants. In particular, the invention provides methods and compositions that allow, for the first time, virus-induced gene silencing in rice. The invention is significant in that prior techniques were not available for rice and because of the major importance of rice to agriculture. The invention therefore provides techniques for the analysis of gene function in rice, as well as in other monocotyledonous species and dicotyledonous plant species.

Description

BACKGROUND OF THE INVENTION [0001] The present invention claims benefit of priority to U.S. Provisional Ser. No. 60 / 479,905, filed Jun. 19, 2003, and U.S. Provisional Ser. No. 60 / 480,705, filed Jun. 23, 2003, the entire contents of each of these applications being hereby incorporated by reference. [0002] 1. Field of the Invention [0003] The invention relates generally to the field of molecular biology. More specifically, it relates to compositions and methods for modulating gene function in plants. [0004] 2. Description of the Related Art [0005] Several technologies have been used to determine plant gene function in vivo. For example, classical breeding of cultivars allows the genetic mapping of various genes. Mutagenesis of plants followed by analysis of progeny identifies gene function through loss of specific phenotypes. Transformation of plants with sequences of unknown function followed by phenotype analysis of progeny is another example of a technology used by research scienti...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K14/08C12N15/82C12N15/86
CPCC07K14/005C12N15/8203C12N2770/14022C12N15/86C12N15/8218
Inventor DING, XINNELSON, RICHARD
Owner SAMUEL ROBERTS NOBLE FOUND
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